FORCE-MEDIATE MEMBRANE FUSION Project summary Membrane fusion is ubiquitous in biology and required for processes as diverse as intracellular vesicle trafficking, viral entry, and fertilization. While the molecules responsible for intracellular vesicle fusion and virus-cell fusion have been studied extensively, much less is known about what controls fusion between two plasma membranes. Over the past decade, a new perspective has emerged that places forces generated by the actin cytoskeleton at the center of the complex sequence of events that results in cell-cell fusion. However, direct evidence of force-mediated membrane fusion is missing due to the difficulty of controlling mechanical force using genetics or biochemistry, leaving unanswered basic questions about the role of force in cell-cell fusion: How much force is needed? Is force necessary to clear proteins and form contact sites? Does force introduce membrane defects and/or promote fusogen activity? In this project, we propose to use in vitro membrane reconstitution combined with high-resolution optical and force microscopy to test the role of external forces on membrane fusion. We will focus on Epithelial fusion failure (Eff-1) protein from C. elegans, a recently- identified cell-cell fusogen whose fusogenic activity is associated with actin assembly in vivo. We hypothesize that fusogens and other proteins that destabilize the plasma membrane are needed to lower the energy barrier to fusion such that the actin cytoskeleton can complete the task, potentially providing a means to restrict cell-cell fusion to times and locations where force and fusogens act in concert. Using reconstituted membrane interfaces as well as live cells, we will quantify fusion efficiency and dynamics using combined fluorescence and force microscopy, and we will compare results with Eff-1 cell-cell fusogen to better-known intracellular and viral fusogens. The results of this study will provide a new biophysical perspective on cell-cell fusion, reveal the dynamic interplay between fusogens and force during fusion initiation, and complement cell-cell fusion studies in model organisms with mechanistic insight that is only possible from in vitro reconstitution.